The delivery of macromolecules into mammalian cells in vitro and in vivo has enabled new areas of research and offers the potential for powerful new treatment options. Recent research has generated many delivery platforms but these solutions remain limited by scope, potency, and safety. The Liu Group recently reported an engineered superpositively charged green fluorescent protein (+36 GFP) with the ability to deliver nucleic acids into a variety of mammalian cell lines in vitro and to potently deliver protein in vitro and n vivo without toxicity. These results led us to identify a subset of naturally occurring human supercharged proteins (SCPs) with similarly potent but previously unknown cell-penetrating and protein delivery properties. Several of these naturally occurring human supercharged proteins, including HBEGF, c-Jun, and N-DEK, were shown to deliver functional Cre recombinase into mammalian cells in vitro and in live adult mice, suggesting that naturally occurring human SCPs may represent a new class of biological delivery agents with a diversity of important properties such as charge, structure, molecular weight, immunogenicity, stability, and in vivo half-life. Building on this foundation, I propose to apply SCPs to deliver human argininosuccinate synthetase I (AS1) into mammalian cell models and mouse models of Type I citrullinemia. AS1 catalyzes the ATP- dependent conversion of citrulline and aspartate to argininosuccinic acid. Deleterious mutations to AS1 are the major cause of Type I citrullinemia, which is characterized by extremely high serum levels of citrulline and ammonia. Citrullinemia patients must be kept on a highly restricted low-protein diet and generally require a liver transplant. Intravenous injectio of adenovirus encoding AS1 extends life in AS1-deficient mouse pups from ~30 hours to ~16 days. Adenoviral treatment, however, loses efficacy upon development of neutralizing antibodies and is associated with other risk factors as well. Here I propose to systematically address the possibility that the direct delivery of AS1 into hepatocytes using SCPs can rescue the metabolic deficiency in citrullinemia cells both in cell culture and in vivo.